Receptor-mediated endocytosis is a vital cellular process that operates to internalize a variety of ligands. Mammalian reoviruses have a broad host range, infecting most mammals including humans, but disease is usually restricted to the very young. Reoviruses efficiently lyse tumor cells in experimental animals and have shown efficacy in clinical trials for aggressive and refractory human tumors. Reoviruses are nonenveloped double-stranded RNA viruses that enter cells via receptor-mediated endocytosis in a beta1 integrin-dependent manner. The viral binding partners of beta1 integrin that promote viral cell entry, the endocytic sorting mechanisms involved in reovirus internalization, and the role of beta1 integrin in reovirus pathogenesis are not known. Reovirus structural protein Iambda2 contains two putative integrin-binding motifs (IBMs), RGD and KGE, but the function of these motifs in reovirus attachment and entry have not been determined. Using a plasmid-based reverse genetics system developed in our laboratory, we have generated viruses with amino acid substitutions in the IBMs of Iambda2. These otherwise isogenic viruses in conjunction with cell lines and mice carrying mutations in the beta1 cytoplasmic tail will be used to evaluate the role of beta1 integrin in reovirus internalization and disease. Three specific aims are proposed. The first specific aim will determine the role of the Iambda2 IBMs in reovirus attachment, internalization, and infectivity. The second specific aim will define the endocytic sorting mechanisms required for productive reovirus infection. The third specific aim will elucidate the role of beta1 integrin and the Iambda2 IBMs in reovirus pathogenesis in vivo using a mouse model of reovirus disease. Collectively, these studies will provide new insights into mechanisms of beta1 integrin-mediated endocytosis of pathogenic viruses and the role of beta1 integrin in viral pathobiology. Public Health Relevance The goal of the proposed experiments is to determine the function of beta1 integrin in reovirus internalization and pathogenesis. These studies will provide a basic understanding of endocytosis and the role of integrins in microbial disease. Furthermore, this work may provide insights that will enhance the use of reovirus as an oncolytic agent by improving the ability to target the virus to malignant cells. [unreadable] [unreadable] [unreadable]